This R01 revision application answers RFA NOT-OD-09-058 (Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications). This is a revision application for the R01 grant GM076330, titled "The Ketoreduction and Cyclization of Aromatic Polyketide Biosynthesis", whose specific aims were 1. Determine the sequence-structure-function relationship of ketoreductase (KR) that leads to its unique reduction specificity. 2. Determine the sequence-structure-function relationship of aromatase/cyclase (ARO/CYC) that leads to its unique cyclization specificity. 3. Determine the importance of protein-protein interactions on the sequence-structure-function relationship between KR and ARO/CYC. We were pleased to report that our progress towards these three aims has exceeded the proposed timelines. In order to further accelerate the research pace towards understanding polyketide ketoreduction and cyclization, we proposed the following three aims that do not overlap, but rather complementary to the parent R01 aims: AIM 1. Determine the molecular basis of ketoreduction using chemical probes. AIM 2. Determine the molecular basis of aromatic polyketide cyclization using chemical probes. AIM 3. Solve crystal structures of chemically cross-linked multi-domain PKS complexes. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long-term biomedical relevance is that the polyketide research community can apply the sequence-structure-function relationships determined from this proposal to diversify the population of "unnatural" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. The revision will accelerate the tempo of scientific research on polyketide synthase (PKS) and allow for job creation and retention for three graduate students and the hiring of one new postdoctoral researcher, and procuring laboratory equipments to accelerate the tempo of the proposed research. PUBLIC HEALTH RELEVANCE: This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities.